Cis-Acting 5 Hammerhead Ribozyme Optimization for In Vitro Transcription of Highly Structured RNAs

RNA-mediated biological processes usually require precise definition of 5′ and 3′ ends. RNA ends obtained by in vitro transcription using T7 RNA polymerase are often heterogeneous in length and sequence. An efficient strategy to overcome these drawbacks consists of inserting an RNA with known boundaries in between two ribozymes, usually a 5′ hammerhead and a 3′ hepatitis delta virus ribozymes, that cleave off the desired RNA. In practice, folding of the three RNAs challenges each other, potentially preventing thorough processing. Folding and cleavage of the 5′ hammerhead ribozyme relies on a sequence of nucleotides belonging to the central RNA making it more sensitive than the usual 3′ hepatitis delta virus ribozyme. The intrinsic stability of the central RNA may thus prevent correct processing of the full transcript. Here, we present a method in which incorporation of a full-length hammerhead ribozyme with a specific tertiary interaction prevents alternative folding with the lariat capping GIR1 ribozyme and enables complete cleavage in the course of the transcription. This strategy may be transposable for in vitro transcription of any highly structured RNA.